British Journal of Cancer (2003) 88, 1213 1216

& 2003 Cancer Research UK All rights reserved 0007 0920/03 $25.00

www.bjcancer.com

Abnormal Fhit expression is an independent poor prognostic

factor for cervical cancer
S Takizawa1, S Nakagawa*,1, K Nakagawa2, T Yasugi1, T Fujii1, K Kugu1, T Yano1, H Yoshikawa3 and Y Taketani1
1

Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan;
Department of Radiology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan; 3Department of
Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaragi 305-8575, Japan

We analysed the expression of the fragile histidine triad (FHIT) gene in cervical cancer to evaluate its clinical relevance in relation to
human papillomavirus (HPV) infection. A total of 73 women with cervical cancer of stage Ib or more advanced (67 squamous cell
carcionomas, four adenocarcinomas, two adenosquamous carcinomas) were examined for Fhit expression by immunohistochemistry.
They were further analysed for the presence of HPV and its subtype. Abnormal expression of Fhit (absent or reduced Fhit
expression) was observed in 52 cases (71.2%). The high-risk HPV DNAs for cervical cancer, including type 16, 18, 31, 33, 51, 52, 58,
68, were identified in 63 cases (86%). The abnormal Fhit expression was not related to the clinicopathological factors including
histology, tumour stage, and HPV type. Notably, the 5-year survival of patients showing the abnormal Fhit expression was significantly
poorer than those showing normal Fhit expression (64 versus 87%, P 0.035). Interestingly, the mean age of the patients with the
abnormal Fhit expression was significantly less than those with the normal Fhit expression (51.6 versus 58.7 years of age, P 0.027,
students t-test). These data imply that the aberrant Fhit expression could be a poor prognostic factor independent of HPV. In the
light of a high incidence of abnormal Fhit expression in younger patients and HPV as a key player in cervical carcinogenesis, abnormal
Fhit expression may accelerate carcinogenesis in concert with HPV.
British Journal of Cancer (2003) 88, 1213 1216. doi:10.1038/sj.bjc.6600892 www.bjcancer.com
& 2003 Cancer Research UK
Keywords: cervical cancer; Fhit expression; HPV

Human papillomavirus (HPV) infection is a major causal factor for

the carcinogenesis of cervical cancer and it is thought to be an
early event of the multistep development of cervical cancer.
Human papillomavirus infection, however, is identified in about
10% of normal women without any abnormality of cervical
cytology, with a very small portion of women with HPV infection
developing cervical cancer, suggesting that mechanisms other than
HPV infection might be involved in cervical carcinogenesis.
The aberrant transcripts of the fragile histidine triad (FHIT)
gene have been identified in some cancers including cervical
cancer and CINs (Negrini et al, 1996; Ohta et al, 1996; Sozzi et al,
1996; Virgilio et al, 1996; Greenspan et al, 1997; Hendricks
et al, 1997; Hadaczek et al, 1998; Nakagawa et al, 1999; Segawa et al,
1999). Higher incidences of the aberrant FHIT expression in
invasive cervical cancer and high-grade CIN than in low-grade CIN
or the normal cervical epithelium suggest that the alteration of
FHIT expression is involved in cervical carcinogenesis (Nakagawa
et al, 1999). To date, two studies described the abnormal Fhit
expression. For instance, Krivak et al (2001) reported that the
abnormal Fhit was an independent poor prognostic factor for stage
II III patients with cervical cancer of squamous type. In contrast,
Helland et al (2000) described that the abnormal Fhit was
associated with poor prognosis, but not an independent poor

prognostic factor by analysing Fhit expression in cervical cancers

for all stages and histological type. These conflicting results give
rise to the question whether or not the alteration of the FHIT gene
is really the causal event in the cancer development. To address
this, we analysed Fhit protein expression in primary cervical
cancer tissues using the immunohistochemical-staining technique
with special reference to an association of the abnormal Fhit
expression with clinical characters.

MATERIALS AND METHODS

Specimens
A total of 73 cases were enrolled in this study, including 67
squamous cell cancers, four adenocarcinomas, and two adenosquamous cancers. The distribution of the tumour stages is as
follows: stage Ib; 20 cases; stage II; 36 cases; stage III; 13 cases;
stage IV; four cases. The mean age of the cases is 54.8 years (28 82
years). We also examined cervical tissues from women undergoing
hysterectomy due to benign gynaecological conditions. All the
subjects gave informed consent for study and had treatment in the
university of Tokyo hospital in 1987 1997.

Analysis of Fhit expression

*Correspondence: Dr S Nakagawa; E-mail: nakagawas-tky@umin.ac.jp
Received 21 October 2002; revised 24 December 2002; accepted 23
January 2003

Each slide, after being deparaffinised and hydrated, was placed in a

container and covered with 0.01 M sodium citrate buffer (pH 6.0)
and heated in a microwave oven (500 W) four times for 5 min each.

Molecular and Cellular Pathology

Analysis of Fhit expression in cervical cancer

S Takizawa et al

1214
After washing in deionised water, endogenous peroxidase was
blocked with 3% hydrogen peroxide for 5 min. The tissue sections
were further blocked with 10% normal pig serum (NPS) (Kohjin
Bio, Japan) in phosphate-buffered saline (PBS) for 10 min at room
temperature to saturate nonspecific binding sites. Then, they were
incubated with diluted primary antibody, anti-FHIT (ZYMED, San
Francisco, CA, USA) at 1 : 200 in PBS overnight at 41C. For negative
control, the slides were incubated with 10% NPS. The slides were
rinsed in PBS, and incubated with biotinylated F(ab0 )2 fragment of
swine anti-rabbit immunoglobulins (DAKO, Tokyo, Japan) at
1 : 500 in PBS for 30 min at room temperature. The slides were
rinsed in PBS, incubated with peroxidase-conjugated streptavidin
(DAKO, Tokyo, Japan) at 1 : 500 in PBS for 30 min at room
temperature, followed by rinsing in PBS, incubation with DAB
(20 mg 3,30 -diaminobenzidine, tetrahydrochloride) for 5 min at
room temperature, and counterstaining with haematoxylin for
1 min.
The degree of Fhit expression was evaluated semiquantitatively
measuring both intensity (no expression or very weak, 1;
moderate, 2; strong positive, 3) and extent (per cent of positive
staining; o10%, 1, 10 50%, 2, 450%, 3). The score of the
intensity was multiplied by the score of the extent to give the total
score for the immunohistochemical staining of Fhit. The total
score of 4 9 was judged as normal based on observations of
noncancerous tissues of the uterine cervix.

Molecular and Cellular Pathology

Detection and typing of HPV

PCR for the L1 region was carried out using the consensus L1
primers L1C1 (50 -CGTAAACGTTTTCCCTATTTTTTT-30 ) (1 mM),
L1C2 (50 -TACCTAAATACTCTGTATTG-30 ) (0.5 mM), and L1C2M
(50 -TACCCTAAATACCCTATATTG-30 ) (0.5 mM). Other conditions
of the PCR were as described elsewhere (Yoshikawa et al, 1991).
Briefly, the PCR protocol was 40 cycles, each cycle consisting of
1.5 min denaturation (951C), 1.5 min annealing (481C), and 2 min

extension (701C). One-tenth of the reaction was electrophoresed

on a 4% agarose gel and stained with ethidium bromide. Human
papillomavirus types were identified on the basis of the restriction
fragment length polymorphisms (RFLP). The PCR-based assay
(L1-PCR) can type at least 26 registered genital HPVs including
type 6, 11, 16, 18, 30, 31, 33, 34, 35, 39, 42, 43, 44, 45, 51, 52, 53, 54,
55, 56, 59, 61, 66, 68, and 70. This PCR system can detect 0.01 pg of
HPV DNA for all these types except for HPV DNA of types 34, 42,
and 55, of which 0.1 mg DNA is minimally needed for detection. We
used b-actin gene amplification to rule out false-negative results.

Statistical analysis
Survival of patients was analysed by the Kaplan Meier technique.
Comparison of mean age was done by Students t-test. Other
statistical analysis methods conducted in this study were w2 test
and multivariate analysis based on the stratified Cox proportional
hazards model.

RESULTS
The intensity of Fhit expression was judged based on a three-tiered
scale comparing Fhit expression between cancers and normal
tissues, that is, the uterine cervix of unaffected women or
histologically normal tissues adjacent to cancers. Fhit was
expressed diffusely in the cytoplasm of epithelial and stromal cells
in the normal tissues (Figure 1A and B). Of the 73 cases with
cervical cancer, 52 cases (71.2%) showed abnormal expression of
Fhit protein. Abnormal Fhit expression, that is, marked reduction
or an absence of Fhit protein expression, is shown in Figure 1C and
D, respectively. The survival of the patients was compared between
the normal Fhit expression and the abnormal Fhit expression using
the Kaplan Meier technique. The 5-year survival of the abnormal
group was significantly poorer than the normal group (64 versus
87%, P 0.035, Figure 2). The mean age of the abnormal group

Figure 1 Immunohistochemical staining of Fhit protein in cervical cancer. (A, B) Normal Fhit expression in cervical cancer. The Fhit expression scores
were 6 (A) and 9 (B). (C) Absent Fhit expression in cervical cancer. Fhit expression in the cervical cancer tissue was negative (score 0). Note that Fhit is
expressed normally in the normal stromal tissue. (D) Marked reduced Fhit expression in cervical cancer. The Fhit expression score in cancer tissue was 3.
British Journal of Cancer (2003) 88(8), 1213 1216

& 2003 Cancer Research UK

Analysis of Fhit expression in cervical cancer

S Takizawa et al

1215

Figure 2 Survival of cervical cancer patients with normal and abnormal

Fhit expression.

Table 1

Number of patients
Age
p50
450
Histology
Squamous cell cancer
Adenocarcinoma
Adenosquamous cancer

HPV types
16
18
Others
Negative

& 2003 Cancer Research UK

DISCUSSION
At present, only a small body of existing literature has described
the clinical implication of normal Fhit expression in cervical
cancer. The present data seem to be in keeping with a previous
report by Krivak et al (2001) who claimed that abnormal Fhit
expression was a poor prognostic factor in patients whose clinical
stages were restricted to stage II or III. Here, we extended this
finding such that abnormal Fhit expression is an independent poor
prognostic factor for all clinical stages inclusive. In contrast, the
data by Helland et al (2000) failed to demonstrate the abnormal
Fhit expression as an independent poor prognostic factor
unrelated to HPV infection. This disparity may be explained, in
part, by the difference of the breakdown of histology or HPV
subtype examined.
It was reported that Fhit protein expression was markedly
reduced or absent in 67 out of 95 (71%) invasive cancer tissues, 17
out of 33 (52%), and eight out of 38 (21%) in high-grade CIN
tissues associated or unassociated with invasive cancer, respectively (Connolly et al, 2000). Considering this and the findings
from our laboratory, the alteration of FHIT expression could occur
at the stage of high-grade CIN and could be, along with HPV, an
important molecular event pertinent to the progression of invasive
cervical cancer.
Human papillomavirus infection is thought to serve as an
initiator for cervical cancer. It follows that HPV infection occurs
well before the alteration of Fhit expression. On the other hand, the
present study demonstrated the abnormal Fhit expression as a

Fhit expression and clinicopathological characters of 73 cervical cancers in Japanese women

Total

Stage
Ib
II
III
IV

The high-risk HPVs including HPV type 16, 18, 31, 33, 51, 52, 58,
and 68 were identified in 63 cases (86%). The abnormal Fhit
expression was observed in 20 out of the 28 cases positive for HPV
16 (71%), in four out of the nine cases positive for HPV 18 (44%),
in 19 out of the 26 cases infected with the other types of HPV (type
31, 33, 51, 52, 58, and 68) (73%), and in nine out of the 10 cases
without HPV infection (90%). The abnormal Fhit expression was
not related to the detection of HPV or its type. The multivariate
analysis with respect to the clinical stage and the age at onset
revealed that the abnormal Fhit expression remains as an
independent poor prognostic factor (Po0.05).

Abnormal
Fhit expression

Normal
Fhit expression

73

52

21

27
46

23(85%)
29(63%)

4(15%)
17(37%)

0.043

67
4
2

48(72%)
1(25%)
2(100%)

19(28%)
3(75%)
0(0%)

0.289

20
36
13
4

15(75%)
24(67%)
10(77%)
3(75%)

5(25%)
12(33%)
3(23%)
1(25%)

0.528

28
9
26
10

20(71%)
4(44%)
19(73%)
9(90%)

8(29%)
5(56%)
7(27%)
1(10%)

0.201

British Journal of Cancer (2003) 88(8), 1213 1216

Molecular and Cellular Pathology

was significantly younger than the normal group (51.6 years versus
58.7 years, P 0.027). In patients at the age of 50 years or older, the
abnormal Fhit expression was detected in 29 out of 46 (63%). In
contrast, 23 out of 27 cases (85%) had the abnormal Fhit
expression in patients lesser than 50 years of age, the difference
reaching statistical significance (Po0.05, Table 1). We further
analysed the relation between the Fhit expression and the mean
age at onset by dividing patients into four groups according to the
Fhit expression score (Table 2). The mean age at onset showed a
tendency of increase along with the rise of the Fhit expression
score. Then we looked at whether Fhit expression is related to the
clinical staging. The abnormal Fhit expression was observed in 15
out of the 20 cases with stage Ib (75%), in 24 out of the 36 cases
with stage II (67%), in 10 out of the 13 cases with stage III (77%),
and in three out of the four cases with stage IV (75%). There was
no significant statistical difference in the rates of the abnormal Fhit
expression among different clinical stages. We next analysed the
relation between the abnormal Fhit expression and HPV infection.

Analysis of Fhit expression in cervical cancer

S Takizawa et al

1216
Table 2

Fhit expression and age at onset of 73 cervical cancers in Japanese women

Fhit expression (score)

Number of cases
Mean age at onset

Negative (0)

Low (1 3)

Medium (4 6)

High (7 9)

Total

3
42.7

40
52.3

25
57.0

5
67.0

73
54.5

poor prognostic factor independent of HPV infection and its

subtype. Besides, our finding of a higher incidence of the abnormal
Fhit expression in relatively younger women with cervical cancer is
noteworthy. If we assume that the age of HPV infection is
essentially the same in cervical cancer patients with or without
abnormal Fhit expression, the above finding could be interpreted
to suggest a role for the abnormal Fhit expression as an accelerator
of carcinogenesis.
At present, we have shown that clinical courses of cervical
cancer are different depending on HPV subtype (Nakagawa et al,
1996). Here, we propose a new determinant for prognosis of
cervical cancer. The abnormal Fhit expression may accelerate the

neoplastic process initiated by HPV infection. The question of

whether or not the abnormal Fhit expression is involved in the
carcinogenesis of cervical cancer by a mechanism similar to HPV
is still open.

ACKNOWLEDGEMENT
This study was supported by Grant-in-aid for Scientific Research
from the Ministry of Education, Science and Culture, Japan.

Molecular and Cellular Pathology

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